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Abstract
The transcription termination factor rho is found exclusively in bacteria and is essential for the viability of many of these organisms. Composed of six identical 47 kDa subunits, rho assembles into a hexameric ring that binds three ATP molecules with high affinity and three with low affinity. Some bacterial RNA transcripts do not have intrinsic stop signals and require rho for proper termination. Rho binds nascent RNA transcripts that have high cytosine content called rut (rho utilizing termination) sites. Once bound to RNA, rho translocates in the 5′ to 3′ direction fueled by ATP hydrolysis and dissociates the RNA message from the DNA template, terminating transcription. Bicyclomycin (1), a structurally-unique antibiotic, is the only known agent that specifically targets rho in vivo. There are two common themes to our investigations: understanding the mechanism of rho translocation along RNA transcripts and mechanisms of rho inhibition.
We conducted a structure-activity relationship (SAR) study in Chapter 2 to identify bicyclomycin fluorescent probes (BFP). Using BFP 34 in fluorescence resonance energy transfer (FRET) studies, we confirmed the postulated ATP-induced conformational change in rho and quantified 34-rho binding parameters at the three high affinity ATP binding protomers.
In Chapter 3 we developed a novel technique to determine bicyclomycin-rho binding and stoichiometry using isothermal titration calorimetry (ITC) and mass spectrometry (MS). We designed a bicyclomycin analog with an appended aldehyde group (64) that efficiently bound to rho augmented by reversible imine bond formation with a nearby lysine residue. We found 1 and 64 bind to all six rho monomers and despite the ATP induced conformational change and heterogeneity of rho ATP binding all six subunits have the same affinity for the inhibitors. We also co-crystallize 64 with rho and solve the first inhibitor bound structure of rho.
We examined a new class of rho inhibitors in Chapter 4. Bismuth complexes of 2,3-dimercaptopropanol (BAL) possess potent antibacterial activity against a broad range of pathogens with previously unknown mechanism(s) of action. We found that BiBAL in a 3:1 ratio inhibits rho with activity comparable to 1 in vitro and modulates rho activity in vivo .